Heliyon 6 (2020) e04566

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Heliyon
journal homepage: www.cell.com/heliyon

Research article

Process optimization for simultaneous production of cellulase, xylanase and

ligninase by Saccharomyces cerevisiae SCPW 17 under solid state
fermentation using Box-Behnken experimental design
Onyetugo C. Amadi a, b, *, Egong J. Egong a, Tochukwu N. Nwagu a, b, Gloria Okpala a,
Chukwudi O. Onwosi a, Greg C. Chukwu a, Bartholomew N. Okolo a, b, Reginald C. Agu b,
Anene N. Moneke a, b
a
Bioprocess and Fermentation Unit, Department of Microbiology, University of Nigeria, Nsukka, Nigeria
b
Brewing Science and Technology Unit, Department of Microbiology, University of Nigeria, Nsukka, Nigeria

A R T I C L E I N F O A B S T R A C T

Keywords: Multienzyme complex has attracted increased attention in biofuel technology. They offer solutions to effective
Bioinformatics degradation of complex plant material into fermentable sugars. Microorganisms, especially bacteria and fungi, are
Biotechnology well studied for their ability to produce enzymes complex unlike yeast. Yeast strain isolated from mushroom farm
Cell biology
was studied for simultaneous production of cellulase, xylanase and ligninase enzymes using lignocellulose waste
Genetics
Microbiology
as substrates. A response surface methodology (RSM) involving Box-Behnken design (BBD) was used to investi-
Molecular biology gate interaction between variables (moisture content, inoculum size, initial pH, incubation time) that affect
Multienzyme complex enzyme production. Crude filtrate was partially purified and characterised. Yeast strain identified as Saccharo-
Cellulase myces cerevisiae SCPW 17 was finally studied. Evaluation of lignocellulose waste for enzyme complex production
Xylanase revealed corn cob to be most effective substrate for cellulase, xylanase and ligninase production with enzyme
Ligninase activity of 17.63
1.45 U/gds, 29.35
1.67 U/gds and 150.75
2.01 μmol/min respectively. Time course study
Corncorb showed maximum enzyme complex production was obtained by day 6 with cellulase activity of 12.5 U/gds,
Saccharomyces cerevisiae
xylanase 48.3 U/gds and ligninase 90.8 μmol/min. Using RSM involving BBD, maximum enzyme activity was
found to be 19.51
0.32 U/gds, 56.86
0.38 U/gds, 408.17
1.04 μmol/min for cellulaase, xylanase and
ligninase respectively. The developed models were highly significant at probability level of P ¼ 0.0001 and
multiple correlation co-efficient (R2) was 0.9563 for cellulase, 0.9532 for xylanase and 0.9780 for ligninase.
Enzyme complex was stable at varying pH and temperature conditions. Saccharomyces cerevisiae (SCPW 17)
studied produced enzyme complex which can be used for bioconversion of biomass to value-added chemicals.

1. Introduction
Producing renewable fuels and chemicals from lignocellulosic
biomass (which are largely abundant in nature) along the biochemical
conversion route requires the hydrolysis of the polysaccharide compo-
nents of biomass, cellulose and hemicellulose, into their various sugar
constituents. The use of enzymes to catalyse the degradation of cellulose
to glucose and hemicellulose to free sugars has long been considered the
most viable strategy to provide cost-effective bioconversion of biomass to
value-added chemicals. Micro-organisms possess several mechanisms for
lignocellulose deconstruction. Fungi and bacteria especially have the free
enzyme system and the most common mechanisms [1]. Furthermore,
cellulosomes and xylanosomes are complex protein structures for the
degradation of biomass, and are largely produced by anaerobic bacteria
and aerobic fungi. Whereas, the potential to produce cellulase or xyla-
nase or both is not commonly found among yeast strains and very scant
literatures are available. Importantly, the economy of biofuel and
chemical production from lignocellulosic biomass requires the use of
cellulose, hemicellulose and lignin in order to obtain an economically
feasible biomass conversion [2].
Several approaches are being proposed – use of enzyme degradation
of lignocellulose biomass, the use of natural strains with multienzyme
complex [3], enzyme cocktail (mixtures of various enzymes from
different sources) or metabolically engineered microbes like

* Corresponding author.
E-mail address: chioma.amadi@unn.edu.ng (O.C. Amadi).

https://doi.org/10.1016/j.heliyon.2020.e04566
Received 12 March 2020; Received in revised form 15 May 2020; Accepted 23 July 2020
2405-8440/© 2020 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
O.C. Amadi et al. Heliyon 6 (2020) e04566

Table 1. Experimental range, coded values and level of independent variables for the optimization of cellulase and xylanase production.

Factors Symbol Coded Level

-1 0 þ1
Moisture Content (%) X1 75 80 85
pH X2 5 6 7
Inoculum Size (ml) X3 1.5 2.0 2.5
Incubation time (days) X4 5 6 7

Table 2. Experimental range, coded values and level of independent variables for the optimization of ligninase production.

Factors Symbol Coded Level

-1 0 þ1
Moisture Content (%) X1 50 60 70
pH X2 4.0 4.5 5.0
Inoculum Size (ml) X3 2.0 3.0 4.0
Incubation time (days) X4 5 6 7

Saccharomyces cerevisiae, Escherichia coli or Yarowia lipolytica to convert
biomass hydrolysates into target products [4] Enzyme market is very
promising in developed countries, like the US, Western Europe, Japan,
and Canada. In these countries the demand for industrial enzymes is
stable, unlike in developing economies, Asia-Pacific, Eastern Europe,
Africa and the Middle East regions that are recently emerging as
fast-growing markets for industrial enzymes [5].
Hydrolases are the most widely used class of enzymes in the industry
[6]. Enzymes are very well-established products in biotechnology. In the
world market, enzymes for industrial applications are expected to be
worth $6⋅3 billion by 2021 [7]. The cost of enzymes for biofuel appli-
cations alone is envisaged to total $1⋅0 billion in 2020, with a CAGR of
10⋅4%. Again, by 2020, the US and European markets for biofuel en-
zymes are expected to be worth $355⋅7 million and $325⋅2 million,
respectively, both with a CAGR >10% (BBC research 2015). Ligno-
cellulose-degrading enzymes are applied in different fields, including
technical use, food manufacturing, paper bleaching, biofuels, textile in-
dustry, and as tools for research and development [8].
High demand in recent years in the enzyme market highlights a need
for development of novel microbial strains capable of producing high

Table 3. Box–Behnken design matrix with experimental values of cellulase and xylanase activity optimization.

Coded values Response (Cellulase activity U/gds) Response (Xylanase activity U/gds)

Run X1 X2 X3 X4 Observed value Predicted value Observed value Predicted value

1 -1 -1 0 0 14.67 14.48 35.07 37.86
2 -1 þ1 0 0 14.16 13.07 39.19 42.35
3 þ1 -1 0 0 8.88 9.34 44.43 45.55
4 þ1 þ1 0 0 11.70 11.26 41.78 43.27
5 0 0 -1 -1 6.98 5.67 13.55 10.72
6 0 0 -1 þ1 6.22 6.22 31.83 32.46
7 0 0 þ1 -1 9.89 9.26 22.08 25.73
8 0 0 þ1 þ1 16.48 17.16 45.70 52.81
9 -1 0 0 -1 6.46 7.99 15.37 16.02
10 -1 0 0 þ1 13.11 13.98 45.49 44.17
11 þ1 0 0 -1 7.04 7.04 24.58 24.05
12 þ1 0 0 þ1 10.17 8.74 47.23 44.73
13 0 -1 -1 0 7.37 7.78 31.76 31.45
14 0 -1 þ1 0 16.83 16.71 51.09 48.33
15 0 þ1 -1 0 9.48 9.70 30.84 31.75
16 0 þ1 þ1 0 15.60 15.29 51.79 50.25
17 -1 0 -1 0 10.51 10.03 33.70 33.07
18 -1 0 þ1 0 19.51 18.88 51.98 47.33
19 þ1 0 -1 0 6.98 8.14 31.72 33.94
20 þ1 0 þ1 0 12.81 13.82 56.86 55.06
21 0 -1 0 -1 6.34 6.38 18.37 18.70
22 0 -1 0 þ1 11.78 11.17 42.03 40.87
23 0 þ1 0 -1 6.07 7.20 18.83 17.56
24 0 þ1 0 þ1 10.37 10.86 46.97 44.22
25 0 0 0 0 11.39 11.10 44.89 41.12
26 0 0 0 0 11.03 11.10 40.01 41.12
27 0 0 0 0 10.88 11.10 38.47 41.12

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O.C. Amadi et al. Heliyon 6 (2020) e04566

Table 4. Box–Behnken design matrix of ligninase activity optimization.

Coded values Response

Run X1 X2 X3 X4 Ligninase activity (μmol/min)

Observed Predicted
1 -1 -1 0 0 352.258 341.326
2 -1 þ1 0 0 377.419 381.416
3 þ1 -1 0 0 366.237 362.635
4 þ1 þ1 0 0 360.645 372.132
5 0 0 -1 -1 232.043 237.581
6 0 0 -1 þ1 341.075 330.071
7 0 0 þ1 -1 203.548 215.107
8 0 0 þ1 þ1 338.280 333.298
9 -1 0 0 -1 218.065 206.907
10 -1 0 0 þ1 313.548 318.053
11 þ1 0 0 -1 220.860 218.725
12 þ1 0 0 þ1 304.731 318.260
13 0 -1 -1 0 346.667 364.030
14 0 -1 þ1 0 357.850 366.988
15 0 þ1 -1 0 408.172 401.405
16 0 þ1 þ1 0 394.194 379.201
17 -1 0 -1 0 355.699 361.311
18 -1 0 þ1 0 346.022 353.838
19 þ1 0 -1 0 380.215 369.474
20 þ1 0 þ1 0 366.237 357.700
21 0 -1 0 -1 223.656 217.234
22 0 -1 0 þ1 329.140 323.596
23 0 þ1 0 -1 240.430 243.049
24 0 þ1 0 þ1 343.871 347.368
25 0 0 0 0 360.645 360.828
26 0 0 0 0 353.011 360.828
27 0 0 0 0 368.828 360.828

enzyme titres, including multienzyme complex, enzyme consortiums and
cocktails combining different enzymes from various sources. This will
help to reduce cost whilst achieving complete deconstruction of ligno-
cellulose as well as building a balanced enzymatic mixture for industry
processes. A promising approach is to source natural strains from unex-
ploited environments that abound in Nigeria. Biodiversity environments
in Nigeria provides great opportunity for isolation and characterization
of novel yeast strains with enzymes producing potentials that can be used
to completely deconstruct lignocellulose biomass. The mushroom farm
environment in Nigeria is a typical example for the isolation of novel
microbial strains capable of producing lignocellulases with high titer.
This is more so when Nigeria has in high volumes lignocellulosic mate-
rials arising from various sources such as cassava peel, cassava pulp,
grasses, plantain/banana waste, yam and cassava decay [9, 10]. Success
in obtaining a novel yeast from the local environment will not only have
the advantage of producing products from these waste materials, it will
also help to provide a cleaner environment [9, 10].
The high moisture content from the lignocellulose materials, nutri-
ents and favourable temperature for growth of the mushroom allows
microbial activity to thrive. It is expected that microbes in this habitat
possess the ability to produce lignocellulases that breakdown ligno-
cellulasic biomass which characterize a mushroom farm – hence this
study. To the best of the authors' knowledge, no studies have been carried
out on the production of combined multienzyme – cellulase, xylanase and
ligninase from yeasts from a mushroom farm. Studies reported in liter-
ature include production of xylanase using psychrophilic yeast Crypto-
coccus adeliae and Cryptococcus flavus isolate I-11 [11,12,13]. Gomes et al.
[14], reported cellulolytic and xylanolytic activities from yeast strains
isolated from bromelied tanks of Viresea minarum, with major genera of
Cryptococcus, Fellomyces, Myriangiale and Ocultifer species. They also re-
ported that only very few of the strains could elaborate cellulolytic and
xylanolytic activities simultaneously. Again, Thongekkew et al. [15],
reported of all the yeast strains isolated from various sources some strains
displayed cellulase activity whilst others displayed xylanase activity. For
production of multienzyme complex, Qadir et al. [16], studied co-culture
of two Saccharomyces cerevisae strains, MK-157and MK-118 to produce
endoglucanase, β-glucosidase and xylanase.
In this paper, we present the results of a novel study where simulta-
neous production of cellulase, xylanase and ligninase was achieved by a
single yeast strain (Saccharomyces cerevisiae SCPW 17) isolated from a
mushroom farm using lignocellulosic waste as substrates. Several factors
such as cultivation conditions and source of nutrient will affect the
production of these enzymes by microorganisms. Optimization of the
process parameters for enhanced production of these enzymes was also
studied and statistically tested using response surface (RSM) method [17,
18, 19]. RSM is a statistical tool for determining optimum conditions of a
process response variables and evaluation of the correlations between a
group of controlled experiments and observing the results of one or more
selected variables [17, 18, 19]. It also brings to bear an alternative
method of optimization where the interactions between all the variables
are taken into consideration and estimate of their combined effect is
expounded. We therefore extended the study to include variables such as
moisture content, initial pH, inoculum size, and incubation time as the
independent variables while the dependent variables (response) were
cellulose, xylanase and ligninase production.
2. Materials and methods
2.1. Soil sample collection
Soil samples were collected from a mushroom farm in Yala Local
Government Area of Cross River State, Nigeria. The samples were

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O.C. Amadi et al. Heliyon 6 (2020) e04566

Table 5. Analysis of variance (ANOVA) test for Box–Behnken design Cellulase.

Source DF Adj SS Adj MS F-Value P-Value

Model 14 334.134 23.867 18.78 0.000
Linear 4 248.202 62.051 48.81 0.000
A-Moisture content 1 36.192 36.192 28.47 0.000
B-pH 1 0.190 0.190 0.15 0.706
C-Inoculum 1 158.268 158.268 124.50 0.000
D-Time 1 53.552 53.552 42.13 0.000
Square 4 60.931 15.233 11.98 0.000
A*A 1 2.193 2.193 1.73 0.214
B*B 1 0.472 0.472 0.37 0.554
C*C 1 5.057 5.057 3.98 0.069
D*D 1 33.200 33.200 26.12 0.000
2-Way Interaction 6 25.001 4.167 3.28 0.038
A*B 1 2.772 2.772 2.18 0.165
A*C 1 2.512 2.512 1.98 0.185
A*D 1 3.098 3.098 2.44 0.144
B*C 1 2.789 2.789 2.19 0.164
B*D 1 0.325 0.325 0.26 0.622
C*D 1 13.506 13.506 10.62 0.007
Error 12 15.254 1.271
Lack-of-Fit 10 15.117 1.512 22.00 0.044
Pure Error 2 0.137 0.069
Total 26 349.389

Model Summary

S R-Sq R-Sq (adj) R-Sq (pred)

1.12747 95.63% 90.54% 74.99%

aseptically collected and placed in sterile sample bottles, properly days at 28
2  C to isolate yeast colonies [20]. The morphologically
labelled, and transported on ice packs to the Microbiology Laboratory of different yeast strains were selected and transferred onto YPDA slants
the University of Nigeria, Nsukka within 24 h of collection. and maintained at 4  C for further studies.

2.2. Strain isolation and inoculum preparation
For initial isolation, appropriate dilutions of soil samples were made
and aliquots inoculated onto agar plates, with media composition (g/L):
NaNO3 - 2.0; KH2PO4 - 1.0; MgSO4.7H2O - 0.5; KCl 0.5; Proteose peptone
- 2.0; Agar - 20, and 0.5% carboxymethyl cellulose (CMC) and xylan was
added respectively. For ligninase, basal medium (LBM), comprised (g/L):
KH2PO4 – 1.0; Yeast Extract - 0.01; C4HI2N2O6- 0.5; CuSO4.5H2O - 0.001;
MgSO4.7H2O - 0.5; Fe2(SO4)3- 0.001; CaCl2.2H2O - 0.01 and
MnSO4.H2O- 0.001. The LBM was supplemented with 0.25 % w/v lignin
and 1.6 % w/v agar and autoclaved. This was supplemented with 10 mL
of a separately sterilized 20 % w/v aqueous glucose solution. A 100 μg/
ml chloramphenicol was added, adjusted to pH 6.0 and incubated for 3–5
2.3. Qualitative (plate) screening of yeast isolates for lignocellulase
production
The yeast isolates were screened qualitatively for their ability to
produce cellulase, xylanase and ligninase simultaneously using CMC,
xylan as well as lignin agar plates to detect their individual cellulase,
xylanase and ligninase activity respectively. Cellulase and xylanase ac-
tivity was carried out according to the method of Srilakshmi et al. [21],
with slight modifications. After 3 days of incubating the plates at 28
2

C, all the plates were flooded with 15 mL of 0.1% Congo red staining
solution for 15 min and distained with 1M solution of NaCl. Isolates with
halo zone from the point of spot inoculation of the culture outwards
denotes substrate (CMC or Xylan) degradation as a result of the enzymes

Figure 1. Phylogenic tree showing relationship of Saccharomyces cerevisiae strain SCPW 17 among the Saccharomyces genus.

4
O.C. Amadi et al.
Figure 2. Cellulase, xylanase and ligninase activity of different carbon sources
by solid state fermentation using Saccharomyces cerevisiae SCPW 17 with
bambara meal as nitrogen source. Key: OB-opete bagasse; SCB-sugar cane
bagasse; CC-corn cob; UP-ugu pod; RH-rice husk.
produced by these yeast isolates. A yellow-opaque zone around a colony
indicates degradation of the substrate and red colour shows
non-degraded substrate. To test for lignin degradation after 3–5 days
incubation in the dark, the LBM plates were flooded with a 1 % w/v
aqueous solution of FeCl3 and K3 (Fe(CN)6) prepared freshly before use.
Phenols in undegraded lignin will stain blue-green, with clear zones
around colonies indicating oxidation of phenolic components [22]. “The
zone of clearance was measured and colonies with wider diameter of
clearance were selected for further studies”.
2.4. Quantitative screening of yeast isolates for lignocellulase production
2.4.1. Submerged fermentation (SmF)
The yeast isolates with wider diameter were also assayed by quanti-
tative method for their cellulose, xylanase and ligninase activity under
submerged fermentation conditions. The isolates were grown in medium
containing the following (g/L): NaNO3-2.0; KH2PO4-1.0; MgSO4.7H2O-
0.5; KCl-0.5; Proteose peptone-2.0; CMC-5.0 for cellulase assay, Xylan-
5.0 for xylanase assay and Lignin for ligninase was added respectively.
Then 50 ml of the medium (pH 6.0) in a 250 mL Erlenmeyer flask was
autoclaved at 121  C for 15 min, cooled, inoculated with 1.0 mL (1.42 
107 CFU/ml) of 24 h old culture and incubated at 28
2  C. Samples were
analyzed after every 24 h for 7 days. Extraction of crude enzyme from
fermentation broth was done by centrifugation at 7,000  g for 30 min
and the clear supernatant (crude enzyme) was used for cellulase and
xylanase assay using DNSA (dinitrosalicylic acid) method [23]. Isolates
with high cellulase activity were also assayed for their xylanolytic ability
and vice versa as well as ligninase. The isolate with most efficient
cellulase, xylanase and ligninase producing ability was characterized by
the amplification of the internal transcribed spacer (ITS1 and ITS2) re-
gions as a means of identification. Ribosomal RNA (r-RNA) sequencing
method and Sanger sequencing of the amplicons was done at the Core
DNA Services Laboratory 188 of the University of Calgary.
2.4.2. Solid state fermentation (SSF)
For the initial study different local agro-waste materials such as corn
cob (CC), sugar cane bagasse (SCB), opete bagasse (OB), rice husk (RH),
ugu pod (UP), were assessed for their potential as substrate for cellulase,
xylanase and ligninase production under solid state fermentation (SSF).
Fermentation was carried out in 250 ml Erlenmeyer flasks that contain
10g of dry weight of each of the above named substrates having particle
size of 425 μm. To determine the effect of particle size on enzyme pro-
duction. The Endecotte machine was used to separate these particles into
different sizes (300 μm, 425 μm, 710 μm and 800 μm). This was moist-
ened at 80% level using the mineral salt medium solution (g/L: NaNO3-
2.0; KH2PO4-1.0; MgSO4.7H2O-0.5; KCl-0.5; bambara meal-2.0) as the
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Heliyon 6 (2020) e04566
moistening agent. The initial pH of the nutrient solution was kept at 6.0
before sterilization. The flasks were sterilized at 121  C for 30 min to kill
all autochthonous microbes present in the sample, allowed to cool and
inoculated with 1.0 mL of isolate suspension (containing 107 CFU/g of
dry substrate). The contents of the flasks were thoroughly mixed, incu-
bated at 28
2  C for the 10 days and samples were taken every 24 h for
analysis [24]. The initial moisture content was determined gravimetri-
cally and all liquid added to the flasks was taken into consideration in
calculating the moisture content. All the experiments were carried out in
triplicate.
2.5. Box-Behnken design (BBD) for optimization of fermentation
parameters using response surface methodology (RSM)
Response Surface Methodology (RSM) involving Box-Behnken design
(BBD) was used to determine the interactions between and amongst all
the variables and their combined effect on cellulase, xylanase and lig-
ninase production. A Box-Behnken factorial design with four factors and
three levels was used to optimize the cultural condition. The independent
variables (factors) used for the analysis were moisture ratio (A), pH (B),
inoculum size (C) and incubation time (D). The effect of these variables
on cellulase, xylanase and ligninase production as well as the most
suitable combination that gave the maximum enzyme yield was also
analyzed. Cellulase, xylanase and ligninase activity extracted were taken
as the dependent variable or response (Y). Each of four independent
variables was studied at three different levels designated as low, middle
and high level of each variable coded as 1, 0 and þ1 respectively
(Tables 1 and 2). A BBD with a total of 27 experimental runs shows the
coded and actual values for each of the enzymes in Tables 3, 4, and 5. The
variables were taken at a central code value considered as zero. The in-
cubation temperature was kept constant at 28
2  C throughout the
entire experiments. Thereafter, the contents of all the flaks were analyzed
for cellulase, xylanase and ligninase activity after 6 days of incubation.
The second order polynomial equation below was adopted to study the
effects of the independent variables on the response.
An empirical second order polynomial equation was adopted to find
the effects of independent variables to the response.
Y ¼ β0 þ β1A þ β2B þ β3C þ β4D þ β1,1A2 þ β2,2B2 þ β3,3C2 þ β4,4D2 þ
β1,2AB þ β1,3AC þ β1,4AD þ β2,3BC þ β2,4BD þ β3,4CD
Where:
Y represents cellulase, xylanase and ligninase activity (response),
β0 is the constant term;
β1, β2, β3 and β4are the coefficient of linear terms;
β1,1, β2,2, β3,3 and β4,4are the coefficient of quadratic terms;
Figure 3. Time course study of enzyme production by Saccharomyces cerevisiae
SCPW 17 grown on corn corb at 28  C in SSF. Key: CA - cellulase activity; XA -
xylanase activity; LA - ligninase activity; TP - total protein content of enzyme; U/
gds - unit per gram of dry substrate; - micromole per min; mg/gds - milligram
per gram of dry substrate.
O.C. Amadi et al.
Figure 4. Response surface plots of effect of different variables on enzyme production (a) cellulase (b) xylanase (c) ligninase by S. cerevisae SCPW 17.
β1,2, β1,3, β1,4, β2,3, β2,4 and β3,4 are the coefficient of cross product
terms respectively. The goodness of fit of the polynomial equation
was expressed by coefficient of determination R2 and its statistical
significance level was checked by F-test.
2.6. Analytical methods for enzyme assays
2.6.1. Enzyme extraction
Enzyme extraction was carried out at 24 h intervals during the in-
cubation period. The crude enzyme was extracted by adding 50 mM
citrate buffer (pH 4.8) at 5 mL of buffer/g of fermented substrate and
agitated at 200 rpm for 30 min. The solid material was separated by
passing the slurry through a muslin cloth and Whatman glass microfiber
filter paper. The filtrate was collected, centrifuged at 1500 g for 20 min
to obtain a clear supernatant and was used to analyze for cellulase and
xylanase activity by 3,5-dinitrosalicylic acid (DNS) method described by
6
Heliyon 6 (2020) e04566
Miller [23] while ligninase assay was done according to the method of
Tien and Kirk [25] with modifications.
2.6.2. Enzyme assays
Cellulase and xylanase activity was determined according to the
method of Bailey et al. [26] with slight modifications. This was done by
mixing 0.5 ml of 1% (w/v) carboxymethylcellulose (CMC) and xylan
prepared in 50 mM citrate buffer (pH 4.8) with 0.5 ml of the appropri-
ately diluted enzyme respectively. The enzyme-substrate mixture was
incubated at 50  C for 10 min. Thereafter, 1.5 mL of the 3,5-dinitrosali-
cylic acid (DNS) reagent was added to terminate the reaction. Enzyme
blanks and a control that contained all the reagents were also run
concurrently but in enzyme blank, the reaction was terminated prior to
the addition of enzyme extract whereas in control, distilled water was
added instead. Thereafter, the tubes were placed in boiling water for 10
min, cooled to room temperature in water for stabilization. The released
reducing sugars were determined spectrophotometrically at 540 nm
O.C. Amadi et al. Heliyon 6 (2020) e04566

Table 6. Analysis of variance (ANOVA) test for Box–Behnken design Xylanase.

Source DF Adj SS Adj MS F-Value P-Value

Model 14 3599.01 257.07 17.47 0.000
Linear 4 2785.04 696.26 47.32 0.000
A-Moisture content 1 55.47 55.47 3.77 0.076
B-pH 1 3.69 3.69 0.25 0.626
C-Inoculum 1 938.10 938.10 63.75 0.000
D-Time 1 1787.79 1787.79 121.50 0.000
Square 4 763.99 191.00 12.98 0.000
A*A 1 12.32 12.32 0.84 0.378
B*B 1 0.80 0.80 0.05 0.820
C*C 1 0.46 0.46 0.03 0.863
D*D 1 576.99 576.99 39.21 0.000
2-Way Interaction 6 49.98 8.33 0.57 0.750
A*B 1 11.46 11.46 0.78 0.395
A*C 1 11.76 11.76 0.80 0.389
A*D 1 13.95 13.95 0.95 0.349
B*C 1 0.66 0.66 0.04 0.836
B*D 1 5.02 5.02 0.34 0.570
C*D 1 7.13 7.13 0.48 0.500
Error 12 176.58 14.71
Lack-of-Fit 10 154.11 15.41 1.37 0.494
Pure Error 2 22.47 11.23
Total 26 3775.59

Model Summary

S R-Sq R-Sq (adj) R-Sq (pred)

3.83597 92.32% 89.87% 75.15%

Table 7. Analysis of variance (ANOVA) test for Box–Behnken design Ligninase.

Source DF Adj SS Adj SS F-Value P-Value

Model 14 92146.7 6581.9 38.03 0.000
Linear 4 35520.3 8880.1 51.31 0.000
A-Moisture content 1 108.5 108.5 0.63 0.444
B-pH 1 1844.2 1844.2 10.66 0.007
C-Inoculum 1 277.8 277.8 1.61 0.229
D-Time 1 33289.9 33289.9 192.34 0.000
Square 4 56029.6 14007.4 80.93 0.000
A*A 1 253.0 253.0 1.46 0.250
B*B 1 581.0 581.0 3.36 0.092
C*C 1 235.2 235.2 1.36 0.266
D*D 1 41728.6 41728.6 241.10 0.000
2 Way Interaction 6 596.7 99.5 0.57 0.744
A*B 1 234.0 234.0 1.35 0.268
A*C 1 4.6 4.6 0.03 0.873
A*D 1 33.7 33.7 0.19 0.667
B*C 1 158.3 158.3 0.91 0.358
B*D 1 1.0 1.0 0.01 0.939
C*D 1 165.1 165.1 0.95 0.348
Error 12 2076.9 173.1
Lack-of-Fit 10 1951.8 195.2 3.12 0.267
Pure Error 2 125.1 62.6
Total 26 94223.6

Model Summary

S R-Sq R-Sq (adj) R-Sq (pred)

13.1559 97.80% 95.22% 87.77%

7
O.C. Amadi et al. Heliyon 6 (2020) e04566

Figure 5. Cellulase, xylanase, ligninase activity and total protein at different ammonium sulphate saturation levels, data is presented as mean
SD of the
three replicates.

Figure 6. Effect of different buffers on partially purified cellulase, xylanase, ligninase from Saccharomyces cerevisiae SCPW 17, data is presented as mean
SD of the
three replicates. Keys: SC3-sodium citrate pH3; SC4-sodium citrate pH4; SC5-sodium citrate pH5; SA4-sodium acetate pH4; SA5-sodium acetate pH5; SA6-sodium
acetate pH6; SP6- sodium phosphate pH6; SP7- sodium phosphate pH7; SP8- sodium phosphate pH8; Tris 9- tris buffer pH9.

using of 3,5-dinitrosalicylic acid (DNS) method with glucose and xylose
calibration curve used as standards for cellulase and xylanase activity
respectively [26]. One unit of cellulose and xylanas is defined as the
amount of enzyme that liberates 1 μmol of glucose and xylose equivalents
per minute, respectively under the standard assay conditions.
While the ligninase peroxide (LiP) assay was performed using 2.6 mL
of the reaction mixture containing 1.0 mL citrate buffer (pH 4.5), 1.0 mL
of 4.0mM veratryl alcohol (3,4-dimethoxybenzylalcohol), 500 μL of 1mM
H2O2 and 100μL of the crude enzyme. A blank containing all other re-
agents except the sample was used. Absorbance was read after a 10 min
reaction interval at 427 nm (Molar absorbtivity, ϵ313 ¼ 9300 M1 cm1).
One unit (U) of LiP activity was defined as the amount of enzyme which
converted 1μmole of veratryl alcohol to veratraldehyde under standard
conditions. The experiments were carried out in triplicate and results
reported as mean
standard deviation.
2.6.3. Ammonium sulphate precipitation
Protein precipitation by salting out technique using ammonium sul-
phate (NH4(SO4)2) was carried out with constant gentle stirring [27].
The crude enzyme was partially purified from the culture supernatant, for
the purpose, various ammonium sulphate concentrations, i.e. 40, 50, 60,
70 and 80 % were used for the precipitation of enzyme. This was left
overnight and the precipitate was collected by centrifugation at 10,000
g for 10 min. The precipitate obtained was dissolved in phosphate
buffer (50 mM, pH 8.0) and dialyzed against the same buffer for 24 h. The
precipitates were collected and analyzed for enzyme complex activity.
2.6.4. Effect of pH on enzyme complex activity and stability
The effect of pH on crude filterate containing enzyme complex
(cellulase, xylanase and ligninase) activity was measured in the pH range
of 3–9, using the appropriate buffers at concentration of 100 mM

8
O.C. Amadi et al.
Figure 7. Thermostability of partially purified (A) cellulase, (B) xylanase (C) ligninase (D) total protein from Saccharomyces cerevisiae SCPW 17, data is presented as
mean
SD of the three replicates.
(3.0–6.0, sodium citrate; 4.0–6.0, sodium acetate; 6.0–8.0 sodium
phosphate; 7.0–9.0, Tris) under standard assay conditions. To study
stability as a function of pH, 100 μL of the partially purified enzyme was
mixed with 100 μL of the buffer solutions and incubated at 28  C for 1 h
then aliquots of the mixture were taken to measure the residual enzyme
complex activity (%) under standard assay conditions.
2.6.5. Effect of temperature on enzyme activity and stability
The influence of temperature on activity of enzyme complex was
studied by incubating the reaction mixture at different temperatures (30,
40, 50, 60, 70). The relative enzyme activity was recorded at 1 h interval
during a period of 6 h. The activity of the enzyme was considered as
100% under standard assay conditions.
2.6.6. Protein content of enzyme
The protein content of the enzyme was determined according to
Lowry et al. [28], using bovine serum albumin (BSA) as standard. The
enzyme sample was properly mixed and 0.5 mL of it transferred to a 10
mL glass tube. 0.7 mL of Lowry solution was added, mixed and incubated
for 20 min at room temperature (28  C) in the dark. After 20 min of
incubation, 0.1 mL of dilute Folin reagent was added to each tube and
mixed. This was incubated at room temperature in the dark. The absor-
bance was measured at 550nm against a blank. Standard curve was
prepared using BSA (1 mg/mL) dissolved in distilled water. All readings
were taken in triplicates.
3. Results and discussion
3.1. Screening and identification of lignocellulose producing yeast
Yeast isolated from mushroom farm capable of simultaneously pro-
ducing cellulase, xylanase and ligninase were screened qualitatively, ten
yeast colonies were picked on the basis of the clear zones around the
colonies. Secondary screening on liquid medium was also carried out to
select strains with the potential to produce the desired enzyme complex.
The yeast strain coded X10 which showed maximum enzyme complex
9
Heliyon 6 (2020) e04566
production was selected. This selected yeast was therefore subjected to
further investigations. Gomes et al. [14], reported isolation of yeast
strains, with cellulolytic and xylanolytic activities with major genera
including Cryptococcus, Fellomyces, Myriangiale and Ocultifer. Just few of
these strains could elaborate cellulolytic and xylanolytic activities,
simultaneously. Thongekkaew et al. [15], also described the ability of 61
yeast strains isolated from different environment to produce xylanase
and cellulose activity. The yeast strain under study was identified on the
basis of the amplification of its internal transcribed spacer (ITS1 and
ITS2) regions as Saccharomyces cerevisiae SCPW 17. The phylogenetic tree
displaying the strain relatedness is shown in Figure 1. It is important to
note that in recent times, novel microbial strains are being sort and
developed especially strains with unique phenotypes that can be used for
industry applications. The environments from which microorganisms are
isolated play critical role in the phenotypes they express. A
genotype-by-environment interaction enables organisms to acclimatize
to environmental variation [29, 30]. Many organisms are able to adapt to
an environment and this influences the phenotype they express [31].
Saccharomyces cerevisiae like other microbes produce extracellular en-
zymes, and the yeasts strains isolated from the mushroom soil showed
varying degrees of lignocellulase enzyme activity more importantly lig-
ninase. This could have been conferred mainly because the genes
responsible for expressing hydrolytic enzymes are present in the strain
and the environment as well as nutrient present influenced the strains
ability to express the enzymes. Hence, the reasons for carrying out this
study.
3.2. Evaluation of different substrates for enzyme complex production
under solid state fermentation (SSF)
Different agro-residues were screened for their ability to be used as
substrate for the production of cellulase, xylanase and ligninase enzyme
complex. Lignocellulosic wastes used include, sugar cane bagasse (SCB),
opete bagasse (OB), corn cob (CC), ugu pod (UP), and rice husk (RH). Our
results showed that corn cob was the most effective substrate for cellu-
lase, xylanase and ligninase production with enzyme activity of 17.63

O.C. Amadi et al.
1.45 U/gds 29.35
1.67 U/gds and 150.75
2.01 μmol/min respec-
tively. SCB was the next best substrate (12.45
1.37 U/gds cellulase;
22.48
1.58 U/gds xylanase and ligninase128.14
1.73 μmol/min) as
shown in (Figure 2). The lignocellulose biomass were used directly
without any pre-treatment as substrate by solid state fermentation for
enzyme complex production, which is typical of what is found in the
environment as in the case of mushroom farm. This part of the experi-
ment is very important as pre-treatment would incur additional costs in
industrial processes. It is important to mention that the composition of
lignocellulose can vary and therefore will affect their accessibility to the
yeast for the enzyme production. This in turn could affect to varying
degrees the production of enzyme complex. This notwithstanding, the
yeast still exhibited the ability to utilize the other substrates to produce
the required enzyme complex.
Reports are available in the literature on the production of cellulases
and xylanase from filamentous fungi and bacteria [32, 33, 34] using
crude LC substrates. However, production of cellulase, xylanase is not
very common with yeast, not to mention ligninase. Gomes et al. [14],
reported cellulolytic and xylolytic activity in some yeast strains, while
Qadir et al. [16], reported cellulase and xylanase activity in coculture of
Saccharomyces cerevisiae MK-157 and Candida tropicalis MK-118. None of
these microorganisms are efficient at cellulase, xylanase and ligninase
activities simultaneously as observed in the present study. Multienzyme
complex (MEC) has become appealing in biofuel technology as they
possibly offer solutions to the more effective degradation of complex
plant material into fermentable sugars, microorganisms involved in
carbon cycling produce an array of carbohydrate degrading enzymes,
including cellulase and xylanases. A number of microorganisms produce
these as free extracellular enzymes, while others produce a multi-enzyme
complex (MEC) such as the cellulosome in which several enzymes such as
cellulases and xylanases are combined within a complex. The Saccharo-
myces cerevisiae SCPW 17 isolated from mushroom farm soil produced
cellulase, xylanase and ligninase enzymes simultaneously in all test
substrate although corn cob was the best and was used for further studies.
3.3. Production profile of cellulase, xylanase and ligninase
SSF is an attractive method for the production of value-added prod-
ucts making use of inexpensive agricultural residues such as CC. So far,
simultaneous production of cellulase, xylanase and ligninase under SSF
of CC by yeasts has not been reported. The time course study of enzyme
production by Saccharomyces cerevisiae SCPW 17 grown on corn corb at
28  C in SSF, is shown in Figure 3. Maximum enzyme complex production
was obtained by day 6 with cellulase activity of 12.95
0.76 U/gds,
xylanase 50.79
2.48 U/gds and ligninase 107.46
5.07 U/gds. The
accumulation of these extracellular enzymes by Saccharomyces cerevisiae
SCPW 17 started after 24 h and increased steadily up to 144 h where the
peak production was observed. The peak production of enzymes for most
microorganisms occurs during the exponential growth phase of the
microbe and this has direct effect on the cost of the production process
because a fast growing strain in optimally rich medium is desirable and
gives high enzyme yields. This agrees with the report of Mahalakshmi
and Jayalakshmi [35] that the highest lignocellulase yield is achieved at
the log stage of the growth curve and decreases with decreases in cell
concentration. Several authors have reported the use of corn cob for
lignocellulase production with varying degrees of enzyme activity [35,
36]. Essentially enzyme productions depend on the chemical composi-
tion of the substrate, accessibility and physiochemical association be-
tween its components [37].
3.4. Optimization of culture conditions for lignocellulase production
3.4.1. Box-Behnken design (BBD) for optimization of fermentation
parameters using response surface methodology (RSM)
Response Surface Methodology (RSM) with BBD was applied to
determine the maximum cellulase, xylanase and ligninase yields and to
10
Heliyon 6 (2020) e04566
study the interactive effect of moisture to substrate ratio, initial pH,
inoculum size and incubation time on enzyme production as well as to
derive a statistical model for their effects. The results of the BBD exper-
iments along with the mean observed and predicted responses for
cellulase, xylanase and ligninase revealed that these data are in reason-
able agreement as shown in Tables 3 and 4 respectively. The response Eq.
(1) represents a suitable model for cellulase recovery while Eq. (2) rep-
resents that of xylanase recovery and Eq. (3) shows ligninase recovery.
P ¼ 11.100–1.737A þ 0.126B þ 3.632C þ 2.112D þ 0.641A2 þ 0.297B2 þ
0.974C2 - 2.495D2 þ 0.832AB - 0.792AC - 0.880AD - 0.835BC - 0.285BD þ
1.837CD (1)
P ¼ 41.12 þ 2.15A þ 0.55B þ 8.84C þ 12.21D þ 1.52A2 - 0.39B2 - 0.29C2 -
10.40D2 - 1.69AB þ 1.72AC - 1.87AD þ 0.41BC þ 1.12BD þ 1.34CD (2)
P ¼ 360.83 þ 3.01 A þ 12.40 B - 4.81 C þ 52.67 D - 6.89 A2 þ 10.44 B2 þ
6.64 C2- 88.45 D2 - 7.65 AB - 1.08 AC - 2.90 AD - 6.29 BC - 0.51 BD þ 6.43
CD (3)
Where, P is the square root of predicted response, and A, B, C and D are
the un-coded (real) values of moisture ratio, initial pH, inoculum size and
incubation time respectively. From the RSM results obtained, run number
18 produced the highest cellulase (19.51 U/gds) while run 20 produced
the highest xylanase (56.86 U/gds) activity which represent 40.36% and
23.29% increase from the earlier result obtained (13.90 U/gds and 46.12
U/gds) respectively. Run 15 gave the highest ligninase activity (408.172
U/gds). This shows the importance of optimizing the production pa-
rameters. Three-dimensional response surface plots were constructed
from the developed models (Figure 4a, b, c) and they exhibit the indi-
vidual and interactive effect of the process variables (see supplementary
material, Figures 1a,b, 2a,b, 3a,b). ANOVA was used to find out the ad-
equacy and fitness of the developed model and the results are shown in
Tables 5, 6, and 7. It was found that the developed models were highly
significant at probability level of P ¼ 0.000. The Fisher's values (F-values)
were calculated and found to be (18.78 for cellulase, 17.47 for xylanase
and 38.03 for ligninase) with very low probability value (P ¼ 0.000),
which exhibit a high degree of adequacy of quadratic models [38]. The
regression equation obtained indicated the R2 value of 0.9563 for
cellulase, 0.9532 for xylanase and 0.9780 for ligninase respectively.
These values have satisfactorily demonstrated that the quadratic model
was highly significant and could explain about 95% of the variability in
the enzyme yield. Similarly different authors have reported analysis of
variance having high coefficient of determination (R2) of 0.9978–0.9070
and P < 0.05 of significant level [39, 40, 41, 42].
3.5. Characterization of enzyme complex
There are several reports on purification and characterization of en-
zymes from various microbial sources [43, 44] but for the first time we
report the purification and characterization of culture filtrate with
multienzyme complex from solid state fermentation of LC by Saccharo-
myces cerevisiae SCPW 17. The crude filtrate containing the enzyme
complex (cellulase, xylanase and ligninase) was subjected to different
ammonium sulphate concentrations (40, 50, 60, 70 and 80%) for pre-
cipitation (Figure 5). It was observed that enzyme activity increased with
increase in (NH4)2SO4 concentration, hence 80% (NH4)2SO4 concen-
tration showed better performance for the enzyme complex precipita-
tion. The pH activity and stability of the partially purified enzyme
complex was determined by measuring the enzyme activity at varying pH
values ranging from 3–9 using different suitable buffers. It was observed
that maximum cellulase and xylanase activity was established at pH 5.0,
sodium citrate buffer, while ligninase was at pH 3.0 sodium citrate buffer
(Figure 6). Stability of the enzyme is a very important factor in studying
characteristics, effect of temperature on partially purified enzyme ac-
tivity was recorded over a broad range of temperature (30–70  C). In case
of partially purified cellulase and xylanase were stable at temperatures
O.C. Amadi et al.
30  C–60  C for 4 h of incubation and retained over 80% activity. At
higher temperature values enzyme stability was gradually declined
(Figure 7). However with ligninase stability was maintained for a period
of 4h at temperature range of 30–50 and 80% of the activity was retained.
At 60 and 70  C degrees enzyme stability gradually declined.
4. Conclusion
The continuous search for novel microbial strains capable of enzyme
complex production for various industrial application is on the rise. Many
studies in this quest to produce multi enzyme complex that can be used
for hydrolysis of waste substrates such as lignocellulosic materials have
been studied and reported. Lignocellulose waste materials are abundant
and transforming them into useful products will be an added advantage
to the economy. None of the report has so far produced microorganisms
capable of producing combined cellulase, xylanase and ligninase activity.
The yeast strain Saccharomyces cerevisiae SCPW 17 studied demonstrated
the potential to simultaneously produce cellulase, xylanase and ligninase
using low cost renewable agricultural waste (corn cob) by solid state
fermentation, without any pre-treatment of substrate, this is important as
pre-treatment would incur additional costs in industrial processes. Box
benkan, Response surface methodology (RSM) employed for the
improvement of cellulase, xylanase and ligninase production was suc-
cessful and was found to be an appropriate method for optimizing the
fermentation parameters to obtain maximum enzyme activity. The sta-
bility of the enzyme complex under a wide range of pH and at varied
temperature also showed the potential of Saccharomyces cerevisiae SCPW
17 for applications in various biotechnology industries.
Declarations
Author contribution statement
Onyetugo C. Amadi, Egong J. Egong: Conceived and designed the
experiments; Performed the experiments; Analyzed and interpreted the
data; Contributed reagents, materials, analysis tools or data; Wrote the
paper.
Tochukwu N. Nwagu, Gloria Okpala, Chukwudi O. Onwosi, Greg C.
Chukwu, Bartholomew N. Okolo, Reginald C. Agu, Anene N. Moneke:
Conceived and designed the experiments; Analyzed and interpreted the
data; Contributed reagents, materials, analysis tools or data; Wrote the
paper.
Funding statement
This research did not receive any specific grant from funding agencies
in the public, commercial, or not-for-profit sectors.
Competing interest statement
The authors declare no conflicts of interest.
Additional information
Supplementary content related to this article has been published
online at https://doi.org/10.1016/j.heliyon.2020.e04566.
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